Increased Oocyte Maturation Research Articles

PRP Influences Maturation and Fertilisation of Immature Mouse Oocytes.

In vitro maturation (IVM) of immature oocytes is a valuable method to enhance the rate of mature oocytes available for fertilisation. In the current study, platelet-rich plasma (PRP) was employed in IVM medium of immature oocytes. Harvested germinal vesicle stage oocytes with cumulus cells from female mature BALB/c mice divided into two groups of control and experiment. In the experimental group, GV oocytes matured in the IVM medium supplemented with 5% PRP, while in the control group, GV oocytes matured in the IVM medium without PRP. The percentage of GV, MI, MII and degenerated oocytes, zona pellucida thickness, perivitelline space size, diameter of mature oocytes, gene expression of apoptosis-related factors and subsequent development of matured oocytes were assessed. The PRP group displayed significantly improved outcomes in various parameters, including a higher proportion of MII and fertilised oocytes, cleavage and blastocyst embryos, compared to the control group. Moreover, the thickness of the zona pellucida was significantly lower in the PRP group than in the control group (p < 0.05). Furthermore, the PRP group demonstrated a significant decrease in the expression of transcripts associated with apoptosis (Bax and caspase-3); however, in the PRP group, a substantial increase in the expression of Bcl2l1, an apoptosis inhibitor, was observed when compared to the control group (p < 0.05). In conclusion, addition of PRP to the IVM culture media significantly increased oocyte maturation rate, leading to improved fertilisation and subsequent embryonic development. This enhancement highlights the positive influence of PRP on overall invitro maturation efficiency and early embryonic stages.

Cytokine-Supplemented Maturation Medium Enhances Cytoplasmic and Nuclear Maturation in Bovine Oocytes.

Bovine in vitro oocyte maturation (IVM) is an easy way to obtain oocytes for subsequent assisted reproductive techniques but is inefficient compared to in vivo maturation. Supplementation of three cytokines, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1), or FLI, has increased oocyte maturation and embryo development in multiple species, but studies have not explored the oocyte differences caused by FLI IVM supplementation. This study aimed to assess important nuclear and cytoplasmic maturation events in high-quality oocytes. FLI-supplemented oocytes had a decreased GV (3.0% vs. 13.7%, p < 0.01) and increased telophase I incidence (34.6% vs. 17.6%, p < 0.05) after IVM, increased normal meiotic spindles (68.8% vs. 50.0%, p < 0.001), and an increased nuclear maturation rate (75.1% vs. 66.8%, p < 0.001). Moreover, in metaphase II oocytes, the percentage of FLI-treated oocytes with a diffuse mitochondrial distribution was higher (87.7% vs. 77.5%, p < 0.05) and with a cortical mitochondrial distribution was lower (11.6% vs. 17.4%, p < 0.05). Additionally, FLI-supplemented oocytes had more pattern I cortical granules (21.3% vs. 14.4%, p < 0.05). These data suggest that FLI supplementation in bovine in vitro maturation medium coordinates nuclear and cytoplasmic maturation to produce higher-quality oocytes.

#50 : Effect of Dual vs. Single (hCG) Trigger in Maturation Rate of Oocytes in ART

Background and Aims: Due to its similar molecular structures and biological activities, human chorionic gonadotropin (hCG) is used as a substitute for luteinizing hormone (LH) in ART. The weaknesses of using hCG as a trigger are the possibility of ovarian hyperstimulation syndrome in high responders and lacked FSH activity which promotes LH receptor formation in luteinizing granulosa cells, nuclear maturation, and cumulus expansion. GnRH agonist (GnRHa) reduces the development of OHSS and induces a physiological surge similar to the natural menstrual cycle. Because of shorter amplitude and duration of GnRHa-induced LH surge, GnRHa-alone trigger results in a higher early miscarriage rate. Combining these two agents; dual trigger increases oocyte maturation with reduced OHSS. Here we present reports in our clinic to differentiate the dual vs. hCG trigger results. Method: We retrospectively analyzed the data from 68 patients who had undergone antagonist protocol from January 2021-January 2023. The final oocyte maturation was undertaken with a dual trigger with GnRHa (Buserelin 0.3 mg) combined with hCG (r-hCG 250 [Formula: see text]g) ([Formula: see text] = 33) or hCG alone ([Formula: see text] = 35). Results: The mean age of both groups was 34 years old. The AMH level of the dual group ranged from 0.06 – 7.17 ng/mL, and in hCG alone group ranged from 0.16 – 12.3 ng/mL. Women in both groups got similar amounts of follicles and MII oocytes; dual group (14.8 and 12.72 vs. 14.9 and 13). The retrieval rate of oocytes was identical in both groups (86.6% vs. 89.9%). However, the maturation rate of dual group was slightly lower than the hCG group (77% vs. 82%), but it was statistically insignificant (independent t-test p 0.99). Conclusion: We showed that dual trigger was not superior to hCG-alone trigger in terms of maturation rate of oocytes. Analysis of larger data is needed to show the real advantages.

Anethole supplementation during in vitro maturation increases in vitro goat embryo production in a concentration-dependent manner

During in vitro maturation (IVM) cumulus-oocyte complexes (COCs) are exposed to conditions that can trigger oxidative stress, thus, reducing oocyte maturation and viability. Aiming to mitigate these detrimental conditions, the effects of IVM medium supplementation with anethole have been tested. Anethole, also known as trans-anethole (1-methoxy-4 [1-propenyl]-benzene), is a naturally occurring phenylpropanoid with various pharmacological properties, including antioxidant effects. However, no study has examined anethole effect on goat COCs during IVM. Thus, the aim of this study was to evaluate the effects of different anethole concentrations on oocyte maturation, oxidative stress, and in vitro development of caprine embryos after parthenogenetic activation. Goat COCs were selected and randomly distributed into the following treatments: TCM-199+ medium (control), or TCM-199+ medium supplemented with 30 μg/mL (AN30); 300 μg/mL (AN300) or 2000 μg/mL (AN2000) of anethole. After IVM, part of the COCs was chosen for oocyte viability and chromatin configuration, intracellular reactive oxygen species levels, and mitochondrial membrane potential assessment. Another part of COCs was parthenogenetically activated, and presumptive zygotes were cultured for 7 days. Results demonstrated that anethole at 30 μg/mL increased oocyte maturation and cleavage rates when compared to the other treatments (P < 0.05), as well as oocyte viability and in vitro embryo production when compared to the control treatment (P < 0.05). Additionally, treatment with anethole at 2000 μg/mL decreased oocyte nuclear maturation and cleavage rates when compared to other treatments (P < 0.05) and embryo production if compared to control and AN30 treatments (P < 0.05). Moreover, anethole at 2000 μg/mL increased mitochondrial membrane potential when compared to the other treatments (P < 0.05). In conclusion, anethole exerts a concentration-dependent effect during goat COCs IVM. For a more desirable outcome of oocyte viability and maturation, and in vitro embryo production, the use of anethole at 30 μg/mL is recommended.

FACTORS AFFECTING THE RATES OF OOCYTE MATURATION AND CRYOPRESERVABLE BLASTOCYSTS IN GNRH ANALOG TRIGGER CASES

The objective of our study was to discover the factors which may affect oocyte maturity rate and the number of blastocysts suitable for freezing. Data were collected from Istanbul Memorial Hospital, ART and Reproductive Genetics Center. 2749 gonadotrophin releasing hormone agonist (GnRHa) trigger cycles between 2011 and 2020 were evaluated retrospectively. Linear regression analysis was used for evaluations of risk factors affecting the maturation rate, and the number of frozen blastocysts. Female age, anti-müllerian hormone (AMH), body mass index (BMI). day-2 LH, progesterone (P) and estradiol (e2) levels were recorded. On GnRHa trigger day e2, LH level and, in some cases P levels were recorded. On the day following GnRHa trigger, LH and P levels in cases where the LH level was below 1 IU/L on trigger day were measured. The total gonadotrophine dosage used, the total days of ovarian stimulation (COS) and the total number of follicles on trigger day were recorded. In addition, the use of pretreatment and additional recombinant LH (rLH) were included in the analysis. Linear regression analyses showed that a one unit increase in female age, day 2 LH value and total gonadotropin dosage, increases oocyte maturation rate with a level of 0.147, 0.274, 0.002; respectively and it was determined that a one-unit increases in AMH value and BMI decreased the maturation rate with a level of 0.242 and 0.212 respectively. Furthermore, a one unit increase in AMH, day 2 LH value trigger day e2 value and the total follicule number on trigger day, increases the number of cryopreservable blastocyst number with a level of 0.211, 0.098, 0.001 and 0.067; respectively. Whereas, a one-unit increases in female age, day-2 e2 value and LH value on trigger day, decreases the number of cryopreservable blastocyst number with a level of 0.142 , 0.011 and 0.263 respectively. However, when the trigger day LH levels were subcategorized as >1 IU/L, 1-0.5 IU/L and <0.5 IU/L and analysed, the oocyte maturation rate was found to be significantly lower in cases with an LH level of <0.5 IU/L. However, despite the lower maturation rate, there was a statistically significantly higher number of cryopreservable blastocyst developed compare to other LH groups on trigger day. This was due to the very high number of oocytes retrieved. In GnRHa trigger patients, female age, AMH, BMI, day-2 e2 and LH levels, total gonadotrophin dosage used, total number of follicles above 11mm on trigger day were the parameters correlated with oocyte maturation and cryopreservable blastocyst. When the trigger day LH levels were subcategorized as >1 IU/L, 1-0.5 IU/L and <0.5 IU/L and maturation rate was significantly lower in cases with an LH level of <0.5 IU/L.

Glucose in a maturation medium with reduced NaCl improves oocyte maturation and embryonic development after somatic cell nuclear transfer and in vitro fertilization in pigs.

This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.

Melatonin improves the maturation and developmental ability of bovine oocytes by up-regulating GJA4 to enhance gap junction intercellular communication.

Melatonin (MT) increases oocyte maturation by reducing reactive oxygen species level and enhancing oocyte antioxidant capacity. However, the mechanisms via which MT works are still poorly understood. In the present study, the effects of MT on the maturation rate and development ability of bovine oocytes were investigated. Then, the transcriptome of oocytes treated by MT was sequenced. Finally, the expression of gap junction protein alpha 4 (GJA4) protein and cAMP level were detected in bovine oocytes, and isoprenaline (enhancer of gap junctional intercellular communication (GJIC)) and heptanol (inhibitor of GJIC) were used to investigate the effect of MT on GJIC activity in bovine oocytes. Our results showed that MT significantly improved the maturation, developmental ability and mRNA expression of GJA4 of bovine oocytes. Meanwhile, MT significantly increased GJA4 protein level and cAMP level in bovine oocytes. In contrast to heptanol, both isoproterenol and MT significantly increased GJIC activity, nuclear maturation and the development ability of bovine oocytes. However, MT significantly restored the nuclear maturation and developmental ability of oocytes treated by heptanol. In conclusion, our results showed that MT improves the maturation and developmental ability of bovine oocytes by enhancing GJIC activity via up-regulating GJA4 protein expression in IVM progress.

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice.

The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus-oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

Coenzyme Q10 supplementation of human oocyte in vitro maturation reduces postmeiotic aneuploidies

To evaluate the effect of coenzyme Q10 (CoQ10) supplementation on oocyte maturation rates and postmeiotic aneuploidy rates during invitro maturation (IVM) of human oocytes. Clinical laboratory observation. Hospital and university laboratories. Forty-five patients aged ≥38 years and 18 patients aged ≤30 years undergoing invitro fertilization. The germinal vesicle-stage oocytes and associated cumulus cells were cultured in IVM media for 24-48 hours with or without 50 μmol/L CoQ10. Oocyte maturation rates were determined based on the presence or absence of the first polar body. Postmeiotic aneuploidies were determined using next-generation sequencing analyses of biopsied polar bodies. Oocyte maturation rates, postmeiotic oocyte aneuploidy rates, and chromosome aneuploidy frequencies. In women aged 38-46 years, 50 μmol/L CoQ10 significantly increased oocyte maturation rates (82.6% vs. 63.0%; P=.035), reduced oocyte aneuploidy rates (36.8% vs. 65.5%; P=.020), and reduced chromosome aneuploidy frequencies (4.1% vs. 7.0%; P=.012. In women aged ≤30 years, we failed to demonstrate an effect of CoQ10 on oocyte maturation rates or postmeiotic aneuploidies. CoQ10 supplementation during IVM increased oocyte maturation rates and reduced postmeiotic aneuploidies for older women.

Inhibition of germinal vesicle breakdown using IBMX increases microRNA-21 in the porcine oocyte

BackgroundGerminal vesicle breakdown (GVBD) occurs during oocyte meiotic maturation, a period when transcriptional processes are virtually inactive. Thus, the maturing oocyte is reliant on processes such as post-transcriptional gene regulation (PTGR) to regulate the mRNA and protein repertoire. MicroRNA (miRNA) are a class of functional small RNA that target mRNA to affect their abundance and translational efficiency. Of particular importance is miRNA-21 (MIR21) due to its role in regulating programmed cell death 4 (PDCD4). The objective of this study was to characterize the abundance and regulation of MIR21 in relation to GVBD.MethodsOocytes were collected from aspirated porcine tertiary follicles. Relative abundance of mature MIR21 was quantified at 0, 8, 16, 24, 32, and 42 h of in vitro (IVM) with or without treatment with 3-isobutyl-1-methylxanthine (IBMX).ResultsIBMX increased abundance of MIR21 at 24 h approximately 30-fold compared to control oocytes (P < 0.05), and the induced increase in MIR21 abundance at 24 h was concomitant with premature depletion of PDCD4 protein abundance. To characterize the effect of artificially increasing MIR21 on oocyte competence without inhibiting GVBD, a MIR21 mimic, scrambled microRNA negative control, or nuclease free water was micro-injected into denuded oocytes at 21 h of IVM. The maturation rate of oocytes injected with synthetic MIR21 (63.0 ± 7.5%) was higher than oocytes injected with negative controls (P < 0.05).ConclusionsInhibition of nuclear meiotic maturation via IBMX significantly increased MIR21 and decreased its target, PDCD4. Injection of a MIR21 mimic increased oocyte maturation rate. Our results indicate MIR21 is active and important during meiotic maturation of the oocyte.

Effect of follicle-stimulating hormone on Bligon goat oocyte maturation and embryonic development post in vitro fertilization.

Background and Aim:Bligon goat is a crossbreed between Etawah and Kacang goat. This crossbreed goat is mostly reared by small farmers. In vitro maturation allows female goat (does) contributes toward reproduction despite the fact that the animal has been slaughtered. The aim of this study was to determine the in vitro maturation rate of Bligon goat oocytes supplemented with follicle-stimulating hormone (FSH), and their ability for further embryonic development after in vitro fertilization.Materials and Methods:Experiment was conducted at the Laboratory of Animal Physiology and Reproduction, Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta, using Bligon goat ovaries obtained from local slaughterhouse around Yogyakarta. One thousand five hundred cumulus-oocyte complexes were matured for 24 h in tissue culture medium 199 supplemented with 50 IU/L FSH or without FSH (control). First, matured oocytes were evaluated its morphology based on the expansion of cumulus cells and PB1 extrusion. Next, 600 oocytes were then stained with 1% aceto-orcein to examine maturation based on changes in the configuration of chromosomes and nuclear membrane breakdown. Oocytes were considered mature when they reached metaphase II. To prove the ability of mature oocytes to develop into embryos, 900 oocytes were processed for fertilization in vitro. The data were analyzed using analysis of variance.Results:The results indicated that FSH supplementation significantly increased oocyte maturation rate (65.21±7.26 vs. 43.25±6.23%) as indicated by extrusion of PB1 and homologous chromosome pairing and lined in the equator. The rate of degeneration was lower in the FSH-supplemented medium (3.21±0.25 vs. 10.17±3.15%). The blastocyst stage of oocyte developed embryos was reached by 12.43±2.15% and 22.28±4.86% of the control and treatment groups, respectively.Conclusion:FSH supplementation significantly improves oocyte maturation and yields mature oocytes for future embryo development in vitro.

Effects of vascular endothelial growth factor supplementation and alginate embedding on human oocyte maturation in vitro

Objective: To evaluate whether the use of vascular endothelial growth factor (VEGF) with alginate increases oocyte maturation following in vitro maturation.Methods: This experimental study was performed on 150 immature oocytes (germinal vesicle oocytes) from females who were candidates for assisted reproductive technology. The germinal vesicle oocytes were randomly placed in the control, alginate, and VEGF plus alginate groups. The basic culture medium for oocytes culture (tissue culture medium 199, follicle-stimulating hormone 0.075 IU/mL, and fetal bovine serum 10%) was used in the control, alginate, and VEGF plus alginate groups. For the treatment groups (alginate, and VEGF plus alginate groups), alginate (8%) and VEGF (5 ng/mL) were added to the basic culture medium. After culture, immature oocytes were considered as oocytes unchanged in the nucleus whereas oocytes with a polar body were considered as mature oocytes (metaphase II stage). The mature oocytes in each group were fertilized by intracytoplasmic sperm injection and formed embryos were evaluated by reverse microscope.Results: The oocyte maturation rate (metaphase II) significantly (P < 0.05) increased in the alginate plus VEGF group as compared with the alginate alone and control groups during in vitro maturation. On day 2, the cleavage rates were significantly different in the matured oocytes between the treatment groups and the control group. The percentage of the two-cell stage, four-cell stage and eight-cell embryos was significantly higher in the treatment groups compared with the control group (P <0.05).Conclusions: Supplementation of VEGF with alginate can improve oocyte maturation in culture media. VEGF with alginate may promote the quality of nuclear and cytoplasmic maturation of human oocytes in vitro.

Melatonin protects against defects induced by malathion during porcine oocyte maturation.

Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.

The effects of kinase modulation on in vitro maturation according to different cumulus-oocyte complex morphologies.

Successful production of transgenic pigs requires oocytes with a high developmental competence. However, cumulus–oocyte complexes (COCs) obtained from antral follicles have a heterogeneous morphology. COCs can be classified into one of two classes: class I, with five or more layers of cumulus cells; and class II, with one or two layers of cumulus cells. Activator [e.g., epidermal growth factor (EGF)] or inhibitors (e.g., wortmannin and U0126) are added to modulate kinases in oocytes during meiosis. In the present study, we investigated the effects of kinase modulation on nuclear and cytoplasmic maturation in COCs. Class I COCs showed a significantly higher developmental competence than class II COCs. Moreover, the expression of two kinases, AKT and ERK, differed between class I and class II COCs during in vitro maturation (IVM). Initially, inhibition of the PI3K/AKT signaling pathway in class I COCs during early IVM (0–22 h) decreased developmental parameters, such as blastocyst formation rate, blastomere number, and cell survival. Conversely, EGF-mediated AKT activation in class II COCs enhanced developmental capacity. Regarding the MAPK signaling pathway, inhibition of ERK by U0126 in class II COCs during early IVM impaired developmental competence. However, transient treatment with U0126 in class II COCs increased oocyte maturation and AKT activity, improving embryonic development. Additionally, western blotting showed that inhibition of ERK activity negatively regulated the AKT signaling pathway, indicative of a relationship between AKT and MAPK signaling in the process underlying meiotic progression in pigs. These findings may help increase the developmental competence and utilization rate of pig COCs with regard to the production of transgenic pigs and improve our understanding of kinase-associated meiosis events.

An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield.

Are meiotic and developmental competence of human oocytes from small (2-8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3-5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF can be reduced by fine-tuning of the culture methods. This novel strategy opens new perspectives for safe and patient-friendly ART in patients with PCOS. IVM research at the Vrije Universiteit Brussel has been supported by grants from: the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680); the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen-FWO, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69). The authors have no conflicts of interest.

176 REGULATORY ROLE OF miR-20a DURING BOVINE OOCYTE MATURATION

The role of microRNA in oocyte maturation is mostly associated with optimal turnover of the accumulated maternal transcripts during their growth to allow maturation. MiR-20a is a member of the miR-17–92 cluster, which has been found to be differentially expressed in bovine granulosa cells derived from preovulatory dominant and subordinate follicles. Our recent study showed that miR-20a is involved in the regulation of granulosa cell proliferation, differentiation, and progesterone synthesis by targeting PTEN and BMPR2 genes. Here, we aimed to investigate the role of miR-20a in the bovine oocyte maturation processes. For this, cumulus-oocyte complexes (COC) were aspirated from small antral follicles (2–8 mm in diameter) and cultured in groups of 50 in 400 µL of maturation media (TCM-199 media supplemented with 12% oestrus cow serum and 10 µg/ml Follitropin®) at 39°C in a humidified atmosphere with 5% (vol/vol) CO2 in the air for 22 h. The cumulus cells and oocytes before (germinal vesicle) and after maturation (metaphase II) were mechanically separated in 0.1% hyaluronidase (in TCM-199 media). To study whether the presence of cumulus cells or oocyte has an impact on the miR-20a expression, we cultured oocytectomized cumulus cells and oocytes with and without their companion cells. Moreover, COC were co-cultured with miR-20a mimic, inhibitor, or corresponding controls to investigate the role of this miRNA in oocyte maturation. The total RNA from cumulus cells and oocytes was extracted using miRNeasy® mini kit (Qiagen GmbH, Hilden, Germany). Total RNA from respective samples was reverse transcribed for mRNA and microRNA expression analysis. Quantitative expression analysis was performed using StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA) and subsequent data were analysed using a comparative cycle threshold method. The progesterone released in the spent media was measured using progesterone enzyme-linked immunosorbent assay kit (ENZO Life Sciences GmbH, Loerrach, Germany). Here, we found that miR-20a expression in cumulus cells increased (P &lt; 0.05) during oocyte maturation. Conversely, miR-20a expression in metaphase II stage oocytes was significantly lower (P &lt; 0.001) compared with the germinal vesicle stage. The absence of oocyte cytoplasm resulted in reduced miR-20a expression in cumulus cells. On the other hand, the absent of cumulus cells increased miR-20a expression in oocytes. The miR-20a expression revealed that the microRNA transduction is restricted in the cumulus cells. The overexpression of miR-20a increased oocyte maturation rate (P &lt; 0.05) by 4.8% (as determined by extrusion of the polar body) and the expression of oocyte maturation-related genes (INHBA, MAPK1, PTGS2, PTX3, and EGFR). The progesterone released in spent media of COC co-cultured with miR-20a mimic and inhibitor showed increasing (P = 0.0936) and decreasing (P = 0.0993) trends, respectively. In this study, we also found that miR-20a modulation altered the expression of PTEN and BMPR2 in cumulus cells. In conclusion, the modulation of miR-20a expression in cumulus cells regulates the oocyte maturation and partially involved in the progesterone synthesis by fine-tuning the expression of PTEN and BMPR2 genes.

Interactions between oocytes and cumulus cells during in vitro maturation of porcine cumulus-oocyte complexes in a chemically defined medium: Effect of denuded oocytes on cumulus expansion and oocyte maturation

The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P < 0.05). Removal of oocytes from COCs by oocytectomy allowed the expansion of CCs to some extent, although their expansion ability was lower than that of COCs (P < 0.05). Addition of DOs (COCs to DOs ratio of 9:16) did not improve cumulus expansion and oocyte maturation rates of intact COCs (P > 0.05) but did enhance cumulus expansion of oocytectomized complexes (P < 0.05). Furthermore, removal of CCs before IVM increased oocyte maturation rates compared with COCs (52.3% and 32.9%, respectively) (P < 0.05) and a similar effect was observed in COCs when the gap junction inhibitor carbenoxolone was added to the IVM medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture through gap junctions. On the basis of these findings, future research could investigate if coculture with DOs during IVM is beneficial for fertilization and embryo development.

Recombinant human growth differentiation factor-9 improves oocyte reprogramming competence and subsequent development of bovine cloned embryos.

Previously, we found that oocyte-secreted factors (OSFs) secreted by denuded oocytes during in vitro maturation (IVM) enhance subsequent development of bovine somatic cell nuclear transfer (SCNT) embryos. This treatment requires many oocytes during IVM. Hence, the aim of this study was to investigate whether supplementing with recombinant growth differentiation factor-9 (GDF9), one of crucial OFSs, in oocyte maturation medium could improve developmental competence of bovine oocytes and subsequent development of cloned embryos. Cumulus-oocyte complexes (COCs) from antral follicles of bovine ovaries collected from an abattoir were cultured with (SCNT+GDF9 group) or without (SCNT group) 200 ng/mL recombinant human GDF9 in oocyte maturation medium. After 22 h, metaphase II (MII) oocytes were used for SCNT. The presence of 200 ng/mL GDF9 significantly increased oocyte maturation rates, the cleavage rate, and blastocyst formation rates of bovine cloned embryos. The blastocyst total, inner cell mass (ICM) cell numbers, and ratio of ICM:TE were higher, whereas the rate of apoptosis in bovine cloned blastocysts was lower in the SCNT+GDF9 group than in the SCNT group. The histone modifications at various sites were also different between each group. These results suggest that COCs cultured with recombinant GDF9 in oocyte maturation medium improve oocyte developmental competence and subsequent developmental competence of cloned embryo in cattle.

Studies on enhancing embryo quantity and quality by immunization against inhibin in repeatedly superovulated Holstein heifers and the associated endocrine mechanisms

To investigate the feasibility of improving embryo production in cattle by immunization against inhibin, both in vivo and in vitro experiments were conducted. In two experiments conducted in two autumns, 14 animals aged 14 months were immunized with a recombinant inhibin α subunit protein antigen for four times at monthly intervals, with another 14 animals of the same age served as the controls. Starting from the second immunization, all the heifers received standard superovulation treatment for three sessions, one session per month, each starting 10 days after every antigen administration. Immunization against inhibin increased number of transferable embryos (P<0.05), and high quality Grade A embryos (P<0.01) in each superovulation. Blood concentrations of FSH, estradiol, activin, and also ratio of activin to follistatin concentrations were greater in inhibin immunized than in control animals during the period of superovulatory FSH administration and animal estrous expression. Heifers immunized with inhibin also had greater concentrations of progesterone in the later diestrus period. In the second experiment, the inclusion of anti-inhibin antibody in oocyte IVM medium increased oocyte maturation rate and cleavage rate following IVF (P<0.05). These results demonstrated that inhibition of the adverse effects of inhibin on ovarian follicular development and oocyte maturation improved embryo yield, in both quantity and quality, following superovulation. These results also demonstrate that active immunization against inhibin, in conjunction with the conventional superovulation protocol, can constitute a new technique for consistent improvement of bovine embryo production in vivo; while passive immunization with anti-inhibin antibody can improve embryo production in vitro.

Inhibition of Cysteine Proteinases During In Vitro Oocyte Maturation Improves Development of Porcine Cumulus-Oocyte-Complexes.

Biochemical differences are known to exist between oocytes that give rise to viable blastocysts compared to oocytes that give rise to embryos that are developmentally compromised. Indeed, mRNA for specific proteolytic enzymes (e.g. cathepsin B) have been shown to be more abundant in in vitro matured bovine oocytes that have diminished developmental potential. The effects of the cysteine proteinase inhibitor, E-64, was recently investigated in bovine cumulus-oocyte-complexes (COCs) chosen to represent both poor- and good-quality oocytes. These reports revealed that the addition of E-64 promoted both oocyte maturation and subsequent embryo development, presumably through inhibition of cathepsin B. We sought to determine if similar results would be obtained in a different oocyte/embryo culture system (i.e. for porcine oocytes). Specifically, it was hypothesized that E-64 would promote maturation of lower-quality oocytes within an aspirated pool; E-64 was also predicted to have little influence on normal oocytes. E-64 was added to the maturation media of ‘good’ v. ‘poor’ COCs that were segregated based on morphological characteristics of the oocytes and by the extent of cumulus cell development. To further determine if cathepsin B itself was influencing COCs, the cathepsin B specific inhibitor, CA-074, was also used during in vitro maturation (IVM) of porcine oocytes. In addition, an analysis of cathepsin mRNA abundance was also performed on cDNA obtained from in vitro matured MII oocytes as a model for ‘lower’ quality oocytes and in vivo (IVV) MII oocytes as a model for ‘good’ quality oocytes. A alterations in cathepsin mRNA abundance were also investigated in IVM porcine COCs and denuded oocytes treated with E-64. Addition of E-64 to IVM medium increased the frequency of maturation in MII oocytes. The highest maturation percentages (74%) were seen in treatment groups containing 20μM E-64 compared to those incubated in the presence of 10 μM E-64 (74% v. 53%; P<0.05) or without E-64 (55%; P<0.05; N=1,750 total oocytes). The percent maturation of ‘good’ oocytes cultured in E-64 was significantly higher than ‘good’ oocytes not exposed to E-64 (68% v. 56%; P<0.001). ‘Poor’ oocytes cultured with E-64 also had significantly higher maturation percentages compared to ‘poor’ oocytes cultured without E-64 (43% v. 31%; P<0.0001). Day six blastocysts that arose from E-64 treated IVM oocytes exhibited no significant increase in total cell numbers relative to untreated ones (34.9 v. 29.8; P=0.19). The use of a cathepsin B specific inhibitor, CA-074, during IVM of oocytes showed no significant effect on oocyte maturation. As with previous experiments, the addition of 20μM E-64 increased oocyte maturation percentages (68% v. 50%; P<0.05). Real-time PCR analysis revealed that cathepsin K mRNA was elevated in IVM MII oocytes compared to IVV MII oocytes as well as in IVM COCs in relation to their denuded counterparts. Cathepsins B and L were not significantly different between IVM and IVV MII oocytes, however cathepsin B did show an increase in abundance in IVM MII COCs compared to IVM denuded MII oocytes. These data suggest that improvements in porcine oocyte maturation arising from E-64 treatment may be due to inhibition of multiple cysteine peptidases and not just from the suppression of cathepsin B, as has been suggested in bovine oocytes. Differences between treatments were determined by SAS with post-test by Tukey's Multiple Comparison Test.